Naïve CD8⁺ T cells abound in the BM, crawl rapidly, and respond to blood-borne antigens. (A) Flow cytometry of CD8α⁺ TCRβ⁺ T cells in the BM and spleen of wild-type mice; dead cells were excluded by 7AAD staining. Memory T cells (CD44⁺) and naïve cells (CD44⁻) are visible. Naïve cells represent ∼77% of CD8 T cells in the spleen and ∼50% of CD8 T cells in the BM. Numbers indicate mean percentage ± standard error of the mean (SEM) from 5 mice examined in 2 separate experiments. (B) An extended-focus snapshot (left) taken by 2PM of cranial BM cavity in a live mouse. Shown are CMTMR-labeled CD8⁺ T cells (yellow) and CFP⁺ B cells (blue) transferred into Cx3cr1^gfp/+ mice (where DCs, monocyte, and macrophages appear green). The BM microvasculature was visualized by intravenous injection of Quantum dots (red). As evidenced by motion tracks (right panel) of CD8⁺ T cells (yellow) and B cells (blue), T cells share the same niches as B cells but crawl faster. Bar represents 50 µm. (C) Three-dimensional tracks of individual CD8⁺ T and B cells. For clarity, tracks were assigned a common origin. (D) Average track velocity of transferred CD8⁺ T and B cells. Data points represent individual cells (n of T cells = 83, n of B cells = 55). *P < .0001 (2-tailed t test). Data are from 1 representative experiment out of 3. (E) Flow cytometry of transferred CD8⁺ OT-I T cells retrieved from the spleen and BM of CD45.2⁺ recipient mice. For transfer, T cells from the spleen and LNs of CD45.1⁺ OT-I mice were immunomagnetically enriched for CD8α expression. Injected cells (top) and cells retrieved 4 hours later from the BM and spleen (middle and bottom) were gated for CD45.1⁺ and CD8⁺ expression (left) and examined for CD62L and CD44 expression (right). The percentage of naïve cells remained constant at ∼90%. Numbers indicate mean percentage ± SEM from 5 mice pooled from 2 independent experiments. (F) Extended-focus snapshots with selected cell tracks (white) of CMTMR-labeled naïve CD8⁺ OT-I T cells (yellow) transferred into Cx3cr1^gfp/+ mice before (left) and after (right) intravenous injection of the OVA peptide SIINFEKL. The BM microvasculature was visualized using Quantum dots (red); GFP⁺ cells appear green. Bar represents 50 µm. (G) Instantaneous velocities during the whole imaging session of individual OT-I cells before and after peptide injection. The motion artifact introduced by peptide injection is followed by a marked decrease in cell speed. Data are from 1 representative experiment of 2. SP, spleen.